HPLC

HPLC

originally referred to:

High Pressure Liquid Chromatography
– high pressure to be able to use small particle size to allow
proper separation at reasonable flow rates
PRINCIPLE
 Principle of HPLC:
The principle of HPLC are based on Van Deemter equation which
relates the efficiency of the chromatographic column to the
particle size of the column, molecular diffusion and thickness of
stationary phase.
The Van Deemter Equation is given as

H or HETP = A + B + C υ
υ
where, A represents eddy diffusion
B represents molecular diffusion
C represents rate of mass transfer
υ represents flow rate
 Basic Components of an HPLC
System
1.Pump System.Mobile phase pressures up to 6000 psi are necessary to
achieve reasonable column elution times (~ minutes). Typical flow
rates are 0.1 to 10 mL/minute.
2.Injection System.Used to introduce small samples (0.1 to 500 μL) into
the carrier stream under high pressure.
3.Reservoirs (Solvents).Multiple solvents are necessary for performing
gradient elution's (i.e. changing the polarity of the mobile phase
during a run).
4.Chromatographic Column. Typically 10-30 cm in length containing a
packing of 5-10 μm diameter. Many types of columns are available,
depending on the type of liquid chromatography desired.
5.Detector.Many types are available including UV, IR, refractive index,
fluorescence, conductivity, mass spectrometry, and electrochemical.
Diode array detectors are used when wavelength scans are desired.
 HPLC Basic Instrumentation

Solvent Delivery Detector

Injector
Column

Separation
Mobile Pump
phase

Sample Injection

Data Processor
 HPLC system
• Solvent Reservoir

• Degasser

• Solvent Delivery System (Pump)

• Injector

• Column &oven

• Detectors
• Recorder (Data Collection)
PUMP SYSTEM
HPLC Pump
• Solvent Reservoir
– Usually one or more glass or stainless steel reservoirs each
of which contains 200-1000 ml of solvent
• Isocratic elution - single solvent separation technique
• Gradient elution - 2 or more solvents, varied during
separation
SAMPLE INJECTION SYSTEM
HPLC Autosampler and Injector
 from Pump to Column from Pump to Column

Needle

Vial
LOAD INJECT
Measuring Pump
HPLC Column
 HPLC Detector
UV/Visible Spectrophotometer
 Column

– straight, 15 to 150
cm in length; 2 to 3
mm i.d.
– packing - silica gel,
alumina, Celite
 CHARACTERISTICS OD
DETECTORS
 Have high sensitivity and the same predictable response
 Respond to all solutes, or else have predictable specificity
 Have a wide range of linearity
 Be unaffected by changes in temperature and mobile-phase
flow
 Respond independently of the mobile phase
 Not contribute to extra column band broadening
 Be reliable and convenient to use
 Have a response that increases linearly with the amount of
solute
 Be nondestructive of the solute
 Provide qualitative information on the detected peak
 Have a fast response
Ultraviolet detector cell for
HPLC
• Problems caused by dissolved air(O2, N2)in mobile phase
– Unstable delivery in pump
Degasser
– Bigger noise and large baseline-drift in detector cell

 In order to avoid causing the problems,

mobile phase should be degassed.
– vacuum pumping systems
– distillation system
– a system for heating and stirring the solvents
– sparging system - bubbles an inert gas of low solubility through the
solvent
 Solvent Delivery System
Requirements
– ability to mix solvents and vary polarity of mobile phase during
run
– “unlimited” solvent reservoir
– generation of pressures up to 6000 psi
– flow rates ranging from 0.1 to 10 mL/min
– flow reproducibility’s of 0.5 % or better
– resistance to corrosion by a variety of solvents
– pulse-free output
 %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load

Ready
inject

Rheodyne
Injector

Solvent Delivery System to injector

through pump

Column
through C
pulse
dampener
A B

from solvent to
Ternary Pump reservoir detector
Chromatography II: HPLC
HPLC Waste Collection
– Use a 10 mL volumetric pipet to add 10.00 mL of soft
drink to 10.00 mL of deionized water.
– Mix thoroughly and half fill a HPLC vial with
your sample. Label the vial with your name and the
name of the soft drink.
• Analysis of Results
– Use the four caffeine standards to prepare a
calibration curve (graph) that plots peak area vs.
concentration.
– Draw a “best fit” straight line on your graph.
– Use your calibration curve to determine the
concentration of caffeine in the sample you prepared.
– Use the concentration of caffeine in your sample,
along with the dilution equation, to determine the
concentration of caffeine in the soft drink you used.
HPLC Chromatogram of Standard 1

0.500 x 10-4 M caffeine

HPLC Chromatogram of Standard 2

1.00 x 10-4 M caffeine

• Calculations
Remember, the original soft drink was diluted to prepare your HPLC
sample. The concentration (M) of caffeine in the original soft drink can
be calculated by using the dilution equation:
M1V1 = M2V2

M1V1
M2 =
V2

The concentration (mg caffeine / 500 mL soda) of caffeine in a full can

of your soft drink can be determine using the following equation:

M2 x 194.2 g caffeine x 1000 mg x 0.500 L = mg caffeine

mol caffeine g in bottle
HPLC - Applications